Batzaya Byambaa

Direct 3D bioprinting of perfusable vascular constructs using a blend bioink.

Despite the significant technological advancement in tissue engineering, challenges still exist towards the development of complex and fully functional tissue constructs that mimic their natural counterparts. To address these challenges, bioprinting has emerged as an enabling technology to create highly organized three-dimensional (3D) vascular networks within engineered tissue constructs to promote the transport of oxygen, nutrients, and waste products, which can hardly be realized using conventional microfabrication techniques. Here, we report the development of a versatile 3D bioprinting strategy that employs biomimetic biomaterials and an advanced extrusion system to deposit perfusable vascular structures with highly ordered arrangements in a single-step process. In particular, a specially designed cell-responsive bioink consisting of gelatin methacryloyl (GelMA), sodium alginate, and 4-arm poly(ethylene glycol)-tetra-acrylate (PEGTA) was used in combination with a multilayered coaxial extrusion system to achieve direct 3D bioprinting. This blend bioink could be first ionically crosslinked by calcium ions followed by covalent photocrosslinking of GelMA and PEGTA to form stable constructs. The rheological properties of the bioink and the mechanical strengths of the resulting constructs were tuned by the introduction of PEGTA, which facilitated the precise deposition of complex multilayered 3D perfusable hollow tubes. This blend bioink also displayed favorable biological characteristics that supported the spreading and proliferation of encapsulated endothelial and stem cells in the bioprinted constructs, leading to the formation of biologically relevant, highly organized, perfusable vessels. These characteristics make this novel 3D bioprinting technique superior to conventional microfabrication or sacrificial templating approaches for fabrication of the perfusable vasculature. We envision that our advanced bioprinting technology and bioink formulation may also have significant potentials in engineering large-scale vascularized tissue constructs towards applications in organ transplantation and repair.

Bioprinted Osteogenic and Vasculogenic Patterns for Engineering 3D Bone Tissue

Fabricating 3D large-scale bone tissue constructs with functional vasculature has been a particular challenge in engineering tissues suitable for repairing large bone defects. To address this challenge, an extrusion-based direct-writing bioprinting strategy is utilized to fabricate microstructured bone-like tissue constructs containing a perfusable vascular lumen. The bioprinted constructs are used as biomimetic in vitro matrices to co-culture human umbilical vein endothelial cells and bone marrow derived human mesenchymal stem cells in a naturally derived hydrogel. To form the perfusable blood vessel inside the bioprinted construct, a central cylinder with 5% gelatin methacryloyl (GelMA) hydrogel at low methacryloyl substitution (GelMALOW ) was printed. We also develop cell-laden cylinder elements made of GelMA hydrogel loaded with silicate nanoplatelets to induce osteogenesis, and synthesized hydrogel formulations with chemically conjugated vascular endothelial growth factor to promote vascular spreading. It was found that the engineered construct is able to support cell survival and proliferation during maturation in vitro. Additionally, the whole construct demonstrates high structural stability during the in vitro culture for 21 days. This method enables the local control of physical and chemical microniches and the establishment of gradients in the bioprinted constructs.

Cell adhesion control on photoreactive phospholipid polymer surfaces

Non-invasive and effective cell recovery from culture substrates is important for the passage and characterization of cells. In this study, a photoreactive polymer surface, which uses UV-irradiation to control substrate cell adhesion, was prepared. The photoreactive phospholipid polymer (PMB-PL) reported herein, was composed of a both 2-methacryloyloxyethyl phosphorylcholine (MPC) unit as a cytocompatible unit and methacrylate bearing a photolabile nitrobenzyl group. The PMB-PL polymer was used to coat a cell culture substrate thus affording a photoreactive surface. Surface analysis of the PMB-PL coating indicated a strong photoresponse owing to the sensitivity of the PL unit. Before light exposure, the PMB-PL surface provided cell adhesion. Following UV-irradiation, the PMB-PL coating was converted to a neutral ζ-potential and hydrophilic surface. The photoreactive surface conversion process allowed for the detachment of adhered cells from the PMB-PL surface while maintaining cell viability. This study demonstrates the promise and significance of the PMB-PL photoreactive surface as a method to control cell attachment and detachment for cell function investigation.

Detachment of cells adhered on the photoreactive phospholipid polymer surface by photoirradiation and their functionality.

Regulating the detachment of adhered living cells from a surface is a key technological requirement to obtain specific cells in the field of cellular engineering. Here, we describe dynamic control of cell adhesion and detachment at a photoreactive and cytocompatible phospholipid polymer surface. The surface was prepared using the amphiphilic and water-insoluble substance poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) bearing 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid (PL) groups in its side chain (PMB-PL). On this prepared surface, the photoinduced control of human epidermoid carcinoma cancer cells (A431 cells) and primary murine embryonic fibroblast (PMEF) cells was examined. The PMB-PL surface allowed successful control of living cells adhesion with photoreactivity. The efficiency of cell detachment obtained was approximately 50% of the initial number of adhered cells. The PL groups at the surface provide adhesion points for cells, as evidenced by the fact that after photoreaction of PL groups by photoirradiation, the number of adhered cells on the surface considerably decreased. Additionally, when the polymer surface was re-used after the first photoirradiation, cells did not adhere to it, and low detachment was observed. The functionalities of the cells detached by photoreaction were evaluated. The proliferation rate and morphological changes of cells were as the same as those of cells detached by conventional enzymatic digestion. Moreover, the extracellular expression levels of transmembrane proteins on cells detached by photoirradiation showed no significant difference with those seen on normal cells. From these results, we conclude that the PMB-PL surface is a suitable platform to regulate cell adhesion and detachment via photochemical reaction.

Engineering Photocrosslinkable Bicomponent Hydrogel Constructs for Creating 3D Vascularized Bone

Engineering bone tissue requires the generation of a highly organized vasculature. Cellular behavior is affected by the respective niche. Directing cellular behavior and differentiation for creating mineralized regions surrounded by vasculature can be achieved by controlling the pattern of osteogenic and angiogenic niches. This manuscript reports on engineering vascularized bone tissues by incorporating osteogenic and angiogenic cell-laden niches in a photocrosslinkable hydrogel construct. Two-step photolithography process is used to control the stiffness of the hydrogel and distribution of cells in the patterned hydrogel. In addittion, osteoinductive nanoparticles are utilized to induce osteogenesis. The size of microfabricated constructs has a pronounced effect on cellular organization and function. It is shown that the simultaneous presence of both osteogenic and angiogenic niches in one construct results in formation of mineralized regions surrounded by organized vasculature. In addition, the presence of angiogenic niche improves bone formation. This approach can be used for engineered constructs that can be used for treatment of bone defects.

Visible light crosslinkable human hair keratin hydrogels

Abstract Keratins extracted from human hair have emerged as a promising biomaterial for various biomedical applications, partly due to their wide availability, low cost, minimal immune response, and the potential to engineer autologous tissue constructs. However, the fabrication of keratin‐based scaffolds typically relies on limited crosslinking mechanisms, such as via physical interactions or disulfide bond formation, which are time‐consuming and result in relatively poor mechanical strength and stability. Here, we report the preparation of photocrosslinkable keratin‐polyethylene glycol (PEG) hydrogels via the thiol‐norbornene “click” reaction, which can be formed within one minute upon irradiation of visible light. The resulting keratin‐PEG hydrogels showed highly tunable mechanical properties of up to 45 kPa in compressive modulus, and long‐term stability in buffer solutions and cell culture media. These keratin‐based hydrogels were tested as cell culture substrates in both two‐dimensional surface seeding and three‐dimensional cell encapsulation, demonstrating excellent cytocompatibility to support the attachment, spreading, and proliferation of fibroblast cells. Moreover, the photocrosslinking mechanism makes keratin‐based hydrogel suitable for various microfabrication techniques, such as micropatterning and wet spinning, to fabricate cell‐laden tissue constructs with different architectures. We believe that the unique features of this photocrosslinkable human hair keratin hydrogel promise new opportunities for their future biomedical applications.

Advances in osteobiologic materials for bone substitutes

A significant challenge in the current orthopedics is the development of suitable osteobiologic materials that can replace the conventional allografts, autografts, and xenografts and thereby serve as implant materials as bone substitutes for bone repair or remodelling. The complex biology behind the nanostructure and microstructure of bones and their repair mechanisms, which involve various types of chemical and biomechanical signalling amongst different cells, has set strong requirements for biomaterials to be used in bone tissue engineering. This review presents an overview of various types of osteobiologic materials to facilitate the formation of the functional bone tissue and healing of the bone, covering metallic, ceramic, polymeric, and cell‐based graft substitutes, as well as some biomolecular strategies including stem cells, extracellular matrices, growth factors, and gene therapies. Advantages and disadvantages of each type, particularly from the perspective of osteoinductive and osteoconductive capabilities, are discussed. Although the numerous challenges of bone regeneration in tissue engineering and regenerative medicine are yet to be entirely addressed, further advancements in osteobiologic materials will pave the way towards engineering fully functional bone replacement grafts.

Porous Electrospun Fibers with Self-Sealing Functionality: An Enabling Strategy for Trapping Biomacromolecules

Stimuli-responsive porous polymer materials have promising biomedical application due to their ability to trap and release biomacromolecules. In this work, a class of highly porous electrospun fibers is designed using polylactide as the polymer matrix and poly(ethylene oxide) as a porogen. Carbon nanotubes (CNTs) with different concentrations are further impregnated onto the fibers to achieve self-sealing functionality induced by photothermal conversion upon light irradiation. The fibers with 0.4 mg mL-1 of CNTs exhibit the optimum encapsulation efficiency of model biomacromolecules such as dextran, bovine serum albumin, and nucleic acids, although their photothermal conversion ability is slightly lower than the fibers with 0.8 mg mL-1 of CNTs. Interestingly, reversible reopening of the surface pores is accomplished with the degradation of PLA, affording a further possibility for sustained release of biomacromolecules after encapsulation. Effects of CNT loading on fiber morphology, structure, thermal/mechanical properties, degradation, and cell viability are also investigated. This novel class of porous electrospun fibers with self-sealing capability has great potential to serve as an enabling strategy for trapping/release of biomacromolecules with promising applications in, for example, preventing inflammatory diseases by scavenging cytokines from interstitial body fluids.

Chaotic printing: using chaos to fabricate densely packed micro- and nanostructures at high resolution and speed

Nature generates densely packed micro- and nanostructures to enable key functionalities in cells, tissues, and other materials. Current fabrication techniques, due to limitations in resolution and speed, are far less effective at creating microstructures. Yet, the development of extensive amounts of surface area per unit volume will enable applications and manufacturing strategies not possible today. Here, we introduce chaotic printing—the use of chaotic flows for the rapid generation of complex, high-resolution microstructures. A simple and deterministic chaotic flow is induced in a viscous liquid, and its repeated stretching and folding action deforms an “ink” (i.e., a drop of a miscible liquid, fluorescent beads, or cells) at an exponential rate to render a densely packed lamellar microstructure that is then preserved by curing or photocrosslinking. This exponentially fast creation of fine microstructures exceeds the limits of resolution and speed of the currently available 3D printing techniques. Moreover, we show that the architecture of the microstructure to be created with chaotic printing can be predicted by mathematical modelling. We envision diverse applications for this technology, including the development of densely packed catalytic surfaces and highly complex multi-lamellar and multi-component tissue-like structures for biomedical and electronics applications.